Convergent evolution of berberine biosynthesis

Abstract

Berberine is an effective antimicrobial and antidiabetic alkaloid, primarily extracted from divergent botanical lineages, specifically <i>Coptis</i> (Ranunculales, early-diverging eudicot) and <i>Phellodendron</i> (Sapindales, core eudicot). In comparison with its known pathway in <i>Coptis</i> species, its biosynthesis in <i>Phellodendron</i> species remains elusive. Using chromosome-level genome assembly, coexpression matrix, and biochemical assays, we identified six key steps in berberine biosynthesis from <i>Phellodendron amurense</i>, including methylation, hydroxylation, and berberine bridge formation. Notably, we discovered a specific class of <i>O</i>-methyltransferases (NOMT) responsible for <i>N</i>-methylation. Structural analysis and mutagenesis of PaNOMT9 revealed its unique substrate-binding conformation. In addition, unlike the classical FAD-dependent berberine bridge formation in Ranunculales, <i>Phellodendron</i> uses a NAD(P)H-dependent monooxygenase (PaCYP71BG29) for berberine bridge formation, originating from the neofunctionalization of tryptamine 5-hydroxylase. Together, these findings reveal the convergence of berberine biosynthesis between <i>Coptis</i> and <i>Phellodendron</i> and signify the role of the convergent evolution in plant specialized metabolisms.